Anthrax

It presents information on pathophysiology, frequency, clinical features, microbiological differences between B anthracis and Non-B anthracis bacilli, differentials, diagnosis, treatment, medication, and follow-up.

Listeria monocytogenes-infected phagocytes can initiate central nervous system infection in mice.

Listeria monocytogenes-infected phagocytes are present in the bloodstream of experimentally infected mice, but whether they play a role in central nervous system (CNS) invasion is unclear. To test whether bacteria within infected leukocytes could establish CNS infection, experimentally infected mice were treated with gentamicin delivered by surgically implanted osmotic pumps. Bacterial inhibitory titers in serum and plasma ranged from 1:16 to 1:256, and essentially all viable bacteria in the bloodstream of treated mice were leukocyte associated. Nevertheless, CNS infection developed in gentamicin-treated animals infected intraperitoneally or by gastric lavage, suggesting that intracellular bacteria could be responsible for neuroinvasion. This was supported by data showing that 43.5% of bacteria found with blood leukocytes were intracellular and some colocalized with F-actin, indicating productive intracellular parasitism. Experiments using an L. monocytogenes strain containing a chromosomal actA-gfpuv-plcB transcriptional fusion showed that blood leukocytes were associated with intracellular and extracellularly bound green fluorescent protein-expressing (GFP+) bacteria. Treatment with gentamicin decreased the numbers of extracellularly bound GFP+ bacteria significantly but did not affect the numbers of intracellular GFP+ bacteria, suggesting that the latter were the result of intercellular spread of GFP+ bacteria to leukocytes. These data demonstrate that infected leukocytes and the intracellular L. monocytogenes harbored within them play key roles in neuroinvasion. Moreover, they suggest that phagocytes recruited to infected organs such as the liver or spleen are themselves parasitized by intercellular spread of L. monocytogenes and then reenter the bloodstream and contribute to the systemic dissemination of bacteria.

Measurement of bacterial ingestion and killing by macrophages.

This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.

Leukocyte-facilitated entry of intracellular pathogens into the central nervous system.

Microbes use numerous strategies to invade the central nervous system. Leukocyte-facilitated entry is one such mechanism whereby intracellular pathogens establish infection by taking advantage of leukocyte trafficking to the central nervous system. Key components of this process include peripheral infection and activation of leukocytes, activation of cerebral endothelial cells with or without concomitant infection, and trafficking of infected leukocytes to and through the blood-brain or blood-cerebrospinal fluid barrier.

Dissemination of Listeria monocytogenes by infected phagocytes.

In vitro data suggest that blood-borne Listeria monocytogenes organisms enter the central nervous system (CNS) by direct invasion of endothelial cells or by cell-to-cell spread from infected phagocytes to endothelial cells. However, a role for infected phagocytes in neuroinvasion and dissemination of L. monocytogenes in vivo has not been confirmed experimentally. Experiments described here tested whether L. monocytogenes-infected peripheral blood leukocytes (PBL) circulated in bacteremic mice and could establish organ infection in vivo. A mean of 30.5% of bacteria cultured from whole blood were PBL associated, and microscopy showed that 22.2% of monocytes and 1.6% of neutrophils were infected. PBL-associated bacteria spread to endothelial cells in vitro, indicating their potential for virulence in vivo. To test this possibility, mice were injected intravenously with infected PBL and CFU of bacteria in liver, spleen, and brain were quantified and compared with values for mice injected with broth-grown bacteria and in vitro-infected macrophage cell lines. An inoculum of infected macrophage cell lines led to greater numbers of bacteria in the liver than the numbers produced by a similar inoculum of broth-grown bacteria. In contrast, brain infection was best established by infected PBL. Results of intraperitoneal injection of infected peritoneal cells compared with results of injection with infected J774A.1 cells suggested that unrestricted intracellular bacterial replication within J774A.1 cells contributed to excessive liver infection in those mice. These data show dissemination of intracellular L. monocytogenes and indicate that phagocyte-facilitated invasion has a role in CNS infection in vivo. Heterogeneity with regard to bactericidal activity as well as to other phagocyte characteristics is a critical feature of this mechanism.

Listeria monocytogenes infection and activation of human brain microvascular endothelial cells.

Listeria monocytogenes invasion of human brain microvascular endothelial cells (BMEC) and its role as a stimulus for endothelial cell activation were studied. Binding and invasion of intact BMEC monolayers were independent of the L. monocytogenes inlAB invasion locus. Cytochalasin D abrogated invasion of BMEC, whereas genistein effected only a 53% decrease in invasion, indicating a requirement for rearrangement of actin microfilaments but less dependence on tyrosine kinase activity. L. monocytogenes stimulated surface expression of E-selectin, ICAM-1, and to a lesser extent, VCAM-1, whereas L. monocytogenes prfA- and Deltahly mutants were severely compromised in this respect. Other experiments showed that BMEC infection stimulated monocyte and neutrophil adhesion and that CD18-mediated binding was the predominant mechanism for neutrophil adhesion to infected BMEC under static conditions. These data suggest that invasion of BMEC is a mechanism for triggering inflammation and leukocyte recruitment into the central nervous system during bacterial meningitis.

Listeria monocytogenes rhomboencephalitis with cranial-nerve palsies: a case report.

Listeria monocytogenes rhomboencephalitis is an uncommon complication of L. monocytogenes meningitis. It presents in a typical biphasic pattern characterized by a non-specific prodromal period followed by any combination of asymmetrical, cranial-nerve palsies; cerebellar signs; hemiparesis or hypesthesia; and diminished consciousness. The survival rate is greater than 70% when appropriate antibiotic therapy is initiated early. However, approximately 60 percent of the survivors develop neurological sequelae. We present the case of a 33-year-old woman who developed L. monocytogenes meningitis with subsequent rhomboencephalitis and cranial-nerve palsie, and review the literature on this syndrome.

Listeria monocytogenes virulence factors that stimulate endothelial cells.

Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L. monocytogenes or L. monocytogenes mutants; then surface expression of E-selectin and neutrophil adhesion were measured. The results showed that delta hly and prfA mutants were the most crippled, requiring 100-fold more mutant bacteria than wild-type bacteria for analogous stimulation. By comparison, L. monocytogenes mutants with deletions of actA, inlA, inlB, inlAB, plcA, and plcB resembled their parent strains, and a delta plcA delta plcB mutant displayed decreased intracellular growth rate but only a minor decrease in stimulation of E-selectin or neutrophil adhesion. Other experiments showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but internalization and increased surface E-selectin and intercellular adhesion molecule-1 expression were profoundly inhibited. However, cytochalasin D had no effect on the HUVEC response to stimulation with lipopolysaccharide or tumor necrosis factor alpha. These data suggest that listeriolysin O production by infecting L. monocytogenes contributes to increased expression of surface E-selectin and intercellular adhesion molecule-1, but neither it nor intracellular replication are directly responsible for this event. Nonetheless it is possible that listeriolysin O potentiates the effect(s) of an other molecule(s) that directly triggers this response. Additionally, cellular invasion by L. monocytogenes appears to be critical for initiating the HUVEC response, potentially by providing a signal which results in upregulation of the necessary bacterial genes.

Listeria monocytogenes infection of cultured endothelial cells stimulates neutrophil adhesion and adhesion molecule expression.

Microbial infection of the endothelium with the resultant up-regulation of adhesion molecule expression and stimulated leukocyte adhesion to endothelial cells can promote an inflammatory response. Previous work demonstrated that Listeria monocytogenes can replicate within cultured endothelial cells; thus, we tested whether L. monocytogenes infection of HUVEC stimulated an inflammatory phenotype on these cells. Infection with 10(4) CFU of bacteria increased neutrophil adhesion to HUVEC 40-fold and up-regulated E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. Approximately 80% of neutrophil adhesion to infected HUVEC was blocked by anti-E-selectin mAb, 35% was blocked by anti-CD18 mAb, and anti-vascular cell adhesion molecule-1 mAb was without effect. Microscopy of infected HUVEC monolayers showed that neutrophils bound to infected and uninfected cells and that infected and uninfected HUVEC expressed E-selectin. Interestingly, uninfected HUVEC that bound neutrophils or expressed E-selectin typically were adjacent to infected cells. However, infected monolayers did not produce soluble factors that stimulated E-selectin expression on uninfected cells. Nuclear translocation of the p65 subunit of the transcription factor NF-kappaB accompanied the HUVEC response, and hemolysin secretion appeared critical for stimulating HUVEC. These studies show that L. monocytogenes infection stimulates up-regulation of adhesion molecule expression on endothelial cells, resulting in neutrophil adhesion to them. This response includes induction of an inflammatory phenotype on uninfected cells and may be triggered by listeriolysin O-mediated activation of host response mechanisms. Additionally, cell-to-cell spread of L. monocytogenes throughout the monolayer, without stimulating secondarily infected endothelial cells for neutrophil adhesion, is a possible means of immune avoidance.

Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines.

Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with noninvasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57Bl/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.

Complement receptor type 3 mediates phagocytosis and killing of Listeria monocytogenes by a TNF-alpha- and IFN-gamma-stimulated macrophage precursor hybrid.

Previous work demonstrated that engagement of complement receptor type 3 (CR3) was required for inflammatory peritoneal macrophages to phagocytose and kill the facultative intracellular bacterium Listeria monocytogenes. The experiments described here tested the role of CR3 in phagocytosis and killing of Listeria by a clonal population of TNF-alpha/IFN-gamma-stimulated macrophage precursor hybrids. Stimulation with TNF-alpha and IFN-gamma increased CR3 expression 20-fold and induced a big increase in phagocytic activity. Phagocytosis and killing of Listeria by these cells were inhibited when bacteria were opsonized with complement-depleted serum or by incubation of the macrophages with anti-CR3 mAb. Furthermore, cytokine-stimulated macrophages could not kill Listeria opsonized with heat-inactivated anti-Listeria antiserum, indicating that macrophage receptors which mediate phagocytosis do not necessarily promote bactericidal activity. These data suggest that upregulation of CR3 and CR3-mediated phagocytosis are mechanisms by which TNF-alpha and IFN-gamma stimulate nonphagocytic, nonbactericidal macrophage precursors to kill intracellular bacterial pathogens.

Fluorescence labeling of bacteria for studies of intracellular pathogenesis.

Interactions between intracellular bacterial pathogens and their eukaryotic cellular hosts or targets are often studied with fluorescence-based techniques such as fluorescence microscopy and flow cytometry. We tested whether the intracellular bacterial pathogens L. monocytogenes, M. avium, M. tuberculosis, and S. typhimurium could be labeled by growth in broth containing the fluorochromes carboxy-X-rhodamine (CR), a hydrazine derivative of fluorescein (FH), and Lucifer Yellow CH (LY). Only Listeria were labeled by all three fluorochromes, Salmonella took up only FH, whereas M. avium and M. tuberculosis were labeled by FH and LY. In general, the fluorochromes did not affect bacterial growth or viability, although FH inhibited the growth of M. tuberculosis. Fluorescent Listeria and M. tuberculosis were used to demonstrate that FH- and LY-labeled bacteria were bright enough after phagocytosis by macrophages to distinguish phagocytic from nonphagocytic cells by flow cytometry. To test whether the fluorochromes might alter bacterial interactions with host cells, we measured both phagocytosis of fluorescent Listeria by macrophages and subsequent bacterial replication in these cells. In these experiments, labeled and unlabeled Listeria were phagocytosed similarly by macrophages and were not impaired in their ability to replicate within them. Thus, this method for fluorescence labeling of bacteria is useful for studying physiologic macrophage:bacteria interactions.

Listeria monocytogenes infects human endothelial cells by two distinct mechanisms.

Infection of endothelial cells by bacteria may be an important component of the bacteria's ability to escape host defenses and cause disease. Listeria monocytogenes cause sepsis and central nervous system infection in domesticated animals and immunocompromised humans, suggesting that this bacterium interacts with endothelial cells in a significant fashion. The experiments presented here tested the hypothesis that L. monocytogenes can invade and replicate within human endothelial cells. We found that L. monocytogenes grows readily in umbilical vein endothelial cells and that its intracellular life cycle involves phagosomal escape, F-actin-based motility, and cell-to-cell spread. We found that L. monocytogenes invaded endothelial cells by cell-to-cell spread from adherent mononuclear phagocytes which were previously infected by this bacterium. Interestingly, L. monocytogenes mutants lacking the invasion protein, internalin, bound less well to endothelial cells than did wild-type bacteria in the absence, but not the presence, of serum, and their invasion of endothelial cells was diminished under both conditions. Thus, endothelial cell infection by L. monocytogenes can occur by two distinct mechanisms: direct bacterial invasion of the endothelial cells in an internalin-mediated fashion or cell-to-cell spread from adherent, infected mononuclear phagocytes. These data support the idea that endothelial cell infection by L. monocytogenes is an important event in the pathogenesis of listeriosis.

TNF-alpha and IFN-gamma stimulate a macrophage precursor cell line to kill Listeria monocytogenes in a nitric oxide-independent manner.

Macrophages are important effector cells for resolving infection with the facultative intracellular bacterium Listeria monocytogenes. However, not all macrophages have the ability to kill this organism. Certain factors, such as cytokines, are apparently required for induction of macrophage bactericidal activity. In vivo studies have shown that both TNF-alpha and IFN-gamma play important roles in resistance against Listeria. Yet whether they act directly on macrophages has been difficult to determine, because homogeneous populations of cells that can be induced to express microbicidal activity have not been available. Instead, bactericidal macrophages are typically found in heterogeneous exudates, such as those elicited by inflammatory agents. In this study we show that sequential stimulation with TNF-alpha and IFN-gamma induces the nonphagocytic, nonbactericidal mouse macrophage precursor hybrid cell line W1C3 to phagocytose and kill Listeria efficiently. This provides the first direct evidence that TNF-alpha and IFN-gamma are both necessary and sufficient to induce macrophages to kill Listeria, and that they act directly on macrophages. Data presented here also show that TNF-alpha and IFN-gamma induced the macrophages to produce large amounts of reactive nitrogen intermediates (RNI), but complete inhibition of RNI generation did not decrease bactericidal activity. This indicates that induction of listericidal activity in these cells does not require generation of RNI. Taken together, these findings suggest that TNF-alpha and IFN-gamma act in synergy directly on at least some macrophages to induce them to express listericidal activity in a RNI-independent manner.

Gentamicin kills intracellular Listeria monocytogenes.

The purpose of the experiments described here was to test whether membrane-impermeant antibiotics present in the extracellular milieu could kill bacteria within macrophages. For this, mouse macrophage hybrids and elicited mouse peritoneal macrophages first were allowed to phagocytose the facultative intracellular bacterium Listeria monocytogenes. The cells were incubated with or without gentamicin, and their bactericidal activity was measured. The results show that gentamicin caused normally nonbactericidal macrophages to kill L. monocytogenes. In addition, gentamicin caused listericidal cells to kill significantly more bacteria. To determine whether gentamicin accumulated within macrophages during culture, we tested whether lysates of macrophage hybrids cultured for 72 h in gentamicin-containing medium and then washed could kill Listeria cells. When cultured with 50 to 100 micrograms of gentamicin per ml, but not when cultured with 0 to 5 micrograms of gentamicin per ml, cell lysates were extremely listericidal, demonstrating the presence of intracellular gentamicin. Because gentamicin does not penetrate cell membranes, we hypothesized that it can be internalized by the cell through pinocytosis and can enter the same intracellular compartment as does phagocytosed L. monocytogenes. To test this, macrophages which had phagocytosed L. monocytogenes were incubated with the fluorochrome lucifer yellow to trace pinocytosed medium. About half of the Listeria cells within the macrophages were surrounded by lucifer yellow, indicating delivery of pinocytosed fluid, which could contain antibiotics, to phagosomes containing bacteria. The experiments described here indicate that membrane-impermeant antibiotics can enter macrophages and kill intracellular bacteria. Thus, the use of gentamicin in macrophage bactericidal assays can interfere with the results and interpretation of experiments designed to study macrophage bactericidal activity.

Complement receptor type 3 (CD11b/CD18) involvement is essential for killing of Listeria monocytogenes by mouse macrophages.

Recent work indicated that C receptor type 3 (CR3) mediates most phagocytosis of the facultative intracellular bacterium Listeria monocytogenes by mouse macrophages, which can kill it. In contrast, phagocytosis of Listeria by a population of nonlistericidal macrophages was largely CR3-independent. These findings suggested that CR3 binding during phagocytosis may be important in determining whether a macrophage kills Listeria, or is parasitized by the bacterium. The experiments reported here tested this hypothesis. When phagocytosis and killing were assayed separately, normally listericidal peritoneal macrophages still could phagocytose to some extent, but lost listericidal activity when CR3 was blocked by mAb. Anti-CR3 mAb inhibited killing in a dose-dependent fashion, and at high doses the cells became permissive hosts. Microbicidal function also was inhibited when active C components were absent during phagocytosis and during killing. Because Listeria are confined to phagosomes in listericidal macrophages but escape into the cytoplasm in nonlistericidal macrophages, we tested whether anti-CR3 mAb enhanced phagosomal escape. In fact, escape of Listeria into the cytoplasm was rare in both control and anti-CR3 mAb-treated macrophages. Moreover, electron microscopy of these cells demonstrated dividing intraphagosomal bacteria. Taken together, these results suggest that binding to CR3 during phagocytosis leads to bacterial killing, and that phagocytic pathways engaged when binding to CR3 is blocked do not trigger microbicidal activity. Furthermore, restriction of Listeria to the phagosome in the absence of CR3 engagement is not by itself sufficient for macrophage listericidal activity.

Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3-mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm.

Listeria monocytogenes is a facultative intracellular bacterium that escapes phagocytic vesicles and replicates in the cytoplasm, where it becomes coated with F-actin. Macrophages, important anti-Listeria effector cells, are heterogeneous in their ability to kill Listeria. Complement receptor type 3 (CR3) mediates most phagocytosis of Listeria by listericidal macrophages. Experiments described here tested whether nonlistericidal macrophages also phagocytosed Listeria through CR3 and whether the ability of Listeria to escape into the cytoplasm correlated with lack of listericidal activity. We show here that CR3 mediated an average of 66% of the phagocytosis of serum-opsonized Listeria by listericidal peptone-elicited macrophages but only 35% by nonlistericidal thioglycolate-elicited macrophages. In thioglycolate-elicited macrophages, most Listeria were cytoplasmic and actin coated, whereas in peptone-elicited macrophages most were retained in the phagosome. These results indicate that listericidal and nonlistericidal macrophages phagocytose Listeria through different receptors and that nonlistericidal macrophages allow Listeria to escape into the cytoplasm.

Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.

One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.

Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages.

Listeria monocytogenes is a facultative intracellular bacterium that is phagocytosed by and can proliferate within cells of the mononuclear phagocyte system. However, the receptors used by macrophages to internalize this organism have not been identified. In the experiments described here, the contributions of serum complement component C3 and macrophage complement receptor type 3 (CR3) to opsonization and phagocytosis of L. monocytogenes by mouse inflammatory peritoneal macrophages were studied. An assay which allowed the distinction of adherent versus internalized bacteria was used to show that following mixing of L. monocytogenes with inflammatory macrophages, greater than 95% of the bacteria bound were internalized by these phagocytes. When immunofluorescent antibodies to C3 and immunoglobulin were used, C3 but not immunoglobulin was detected on L. monocytogenes following incubation in normal serum or ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetracetic acid-Mg(2+)-chelated serum. When macrophages were incubated with 5% serum and L. monocytogenes in a standard assay, approximately 80% of the phagocytosis was inhibited by heat-inactivated serum or by the addition of F(ab')2 anti-C3 antibody. The role of macrophage CR3 was demonstrated by the ability of anti-CR3 monoclonal antibody M1/70 to decrease phagocytosis to the same levels as those seen with heat-inactivated serum. These experiments indicated that in the presence of normal serum, L. monocytogenes is phagocytosed by inflammatory macrophages primarily because CR3 on these cells binds to C3 deposited on the bacterial surface.